Campylobacter jejuni causes severe human enteritis. The morbidity associated with this disease is considerable, especially in immunocompromised patients or those with underlying disease. Three potential mechanisms of disease induction have been proposed for this bacterial pathogen: (A) invasion of host cells followed by intracellular replication resulting in damage to cells and tissues deep in the lamina propria and mesenteric lymph nodes; (B) production of toxins, the most notable of which include a potent cytotoxin which adheres to host-cell membranes and effects cytotoxic damage to cells and tissues; a cytolethal distending toxin which induces cytotonic changes in cells and an enterotoxin which modulates intracellular levels of cyclic nucleotides; and (C) combinations of these. Although (C) probably reflects most accurately the pathogenic profile for this agent, mounting evidence suggests that the cytotoxin may play a crucial role in the pathogenic strategy of the organism thereby profoundly affecting the outcome of disease. The purpose of this AREA proposal is to address those issues concerning the nature of the cytotoxin. Two major objectives are defined: (1) to clone those genes encoding the cytotoxic protein and to sequence the genes responsible for expression; (2) to investigate attachment of the cytotoxin to host cells and to obtain a better understanding of the fate of the toxin after binding. The molecular biology procedures to be used for cloning and sequencing are well established. Clones carrying Campylobacter jejuni DNA fragments will be generated using lambdagt11 or lambdaZAPII. Polyclonal antibody specific for the cytotoxin of C. jejuni and the anti-cytotoxin monoclonal antibody, CETPMAb/4 (already developed by us), will be used to screen clones for cytotoxin expression. Should clones prove nonexpressive, then probes will be developed. Fragments of the cytotoxic protein will be characterized using Edman chemistry and automated sequencing procedures to generate oligonucleotide probes for the cytotoxin genes. Nucleotide sequencing of the cytotoxin genes using automated facilities will follow. The location of the cytotoxin in the affected host cell will be examined by fluorometry, cell fractionation techniques and immunoelectron microscopy. In addition, data will be collected concerning the nature of the host-cell membrane receptor responsible for binding the cytotoxin. The overall goal of this work is to clarify the pathogenesis of Campylobacter jejuni induced enteritis. Indeed, the work will provide molecular and genetic data concerning the fundamental nature of the bacterial cytotoxin as well as provide molecular reagents suitable for probing the virulence determinants of this important human pathogen. In addition, the fate of the toxin after the host-cell binding event has occurred will be examined. These studies will supply a basis for further defining the virulence of Campylobacter jejuni and may provide potential and novel methods for control and detection of clinical campylobacteriosis.